Ojective 1: Characterize the mechanisms by which Epag promotes trilineage hematopoiesis. 1.1 Epag evades IFN blockade of signal transduction from c-MPL in human HSPCs. By directly comparing the effect of IFN on human HSPCs in the presence of TPO or Epag in vitro, we made two observations. First, we found that IFNg perturbed TPO-induced signaling pathways in human HSPCs and that Epag could bypass this inhibition, resulting in enhanced progenitor activity and long-term HSPC repopulating potential in the presence of IFNg. Second, we showed that IFNg disrupted the low-affinity interaction between TPO and the extracellular domain of c-MPL, delineating a novel molecular mechanism by which IFNg inhibits TPO signaling in HSPCs. Epag may bypass the inflammatory inhibition of signal transduction by avoiding capture by IFNg in the bone marrow, thus providing an explanation for its clinical efficacy in SAA, despite already elevated levels of TPO in these patients. This work was published (Blood 133:2043-55 (2019)). 1.2 Epag promotes DNA DSB repair in human HSPCs. To assess whether Epag promotes DNA DSB repair in human HSPCs and to identify the pathways involved, CD34+ cells obtained from healthy individuals were first cultured for 24 hours in the presence or absence of Epag and the DNA-PK inhibitor NU7441, exposed to low dose (2Gy) IR, and either transfected with NHEJ or HR DNA DSB repair reporter plasmids or assessed for changes in H2AX phosphorylation (H2AX), an indicator of IR-induced DSBs, at various times after IR. We demonstrated that Epag specifically activates the canonical NHEJ (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Importantly, Epag-mediated DNA repair resulted in enhanced genome stability, survival and function of primary HSPCs, as demonstrated in karyotyping analyses, CFU assays and after transplantation in immunodeficient NSG mice. Eltrombopag may thus offer a new therapeutic modality for the prevention of HSPC injury induced by IR in cancer therapy, and could have implications for the treatment of genome instability syndromes such as FA. This work was published (Exp Hematol 73:1-6 (2019)). Objective 2: Evaluate the safety and efficacy of Epag in subjects with Fanconi anemia. We have initiated a clinical trial to investigate whether Epag may offer a novel therapeutic modality for subjects with FA. Our pre-clinical studies described above indicate that Epag evades blockade of signal transduction from c-MPL induced by inflammatory cytokines. Additionally, we found that Epag enhances DNA repair activity in human HSPCs. Thus, Epag may positively influence two of the main known mechanisms leading to BMF in FA. Study Design. This is a non-randomized, phase II study of Epag given to subjects with FA (NCT03204188). Subjects receive Epag for 6 months. Subjects who cannot tolerate the medication or fail to respond by 6 months are taken off study drug. Subjects who respond at 6 months are invited in the extension phase for an additional 3 years. Eligibility Assessment. Inclusion criteria: (1) Confirmed diagnosis of FA by a biallelic mutation in a known FANC gene and/or by positive chromosome breakage analysis in lymphocytes and/or skin fibroblasts; (2) One or more of the following cytopenias: platelets 30K/L or platelet transfusion dependence in the 8 weeks prior to study entry, ANC 500/L, Hgb 9.0 g/dL or red blood cell (RBC) transfusion dependence in the 8 weeks prior to study entry; (3) Failed or declined treatment with androgens; 4) Age > 4 years. Exclusion criteria: (1) Evidence of MDS or AML; (2) Cytogenetic abnormalities associated with poor prognosis in FA; (3) Known biallelic mutations in BRCA2; (4) Active malignancy or likelihood of recurrence of malignancies within 12 months; (5) Treatment with androgens 4 weeks prior to initiating EPAG. Primary Endpoints. The primary efficacy endpoint is the proportion of drug responders at 6 months. Response to Epag is defined by one or more of the following criteria: (1) Platelets increase by 20K/L above baseline, or platelet transfusion independence; (2) Hgb increase by > 1.5g/dL or a reduction in the units of RBC transfusions by at least 50%; (3) At least a 100% increase in ANC for subjects with a pretreatment ANC of < 0.5 x 109/L, or an ANC increase > 0.5 x 109/L. The primary safety endpoint is the toxicity profile assessed at 6 months using the CTCAE criteria. Enrollment. Two subjects have been enrolled to date. No drug-related adverse events have been observed. Subject #1 (7YO female) did not respond to 6 months of Epag, likely due to limited HSPC reserve in the context of profound cytopenias (ANC = 100/L, Hgb = 6g/dL, Plt = 0K/L). In contrast, subject #2 (49YO female) showed response to Epag at 3 months and will continue on the extension phase of the study. This study will continue in FY20 and is expected to provide important clinical information on safety and efficacy of Epag in subjects with FA. Objective 3: Evaluate the ability of Epag to improve erythropoiesis in Diamond Blackfan anemia. In this study, we investigated whether Epag could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced ROS and increased proerythroblast survival and maturation. To test this possibility, we first established an iPSC model of DBA by reprogramming MNCs from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line. RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTM monolayer-based approach. Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+ primitive erythroid progenitors (P1), CD71+CD45loEPOR- proerythroblasts (P2), and more mature CD71+CD45-EPOR- erythroblasts (P3). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage. Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitors inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 g/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations. Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA.